engleski

Cryopreservation and cryotherapy of grapevine (Vitis vinifera L.)

This study aimed at establishing a cryopreservation protocol for grapevine shoot tips and at testing the efficiency of cryopreservation in eliminating selected grapevine viruses. In vitro cultures of healthy genotypes of eight Croatian autochthonous grapevine cultivars Plavac mali, Maraština, Pošip, Debit, Grk, Lasina, Plavina and Vugava and of virus-infected genotypes of Plavac mali were successfully established. Differences in survival, regrowth and growth parameters were genotype-specific. Infected cultivars were less reactive compared to healthy ones. A PVS2-based cryopreservation protocol was successfully established. Modifications in sucrose preculture conditions and use of PVS2-derived alternative vitrification solutions did not improve growth recovery. By contrast, the physiological state of the plant material played a critical role in cryopreservation. Actively growing buds sampled from single-node microcuttings displayed higher regrowth compared to buds sampled directly on in vitro plantlets. The position of buds on the stem of in vitro mother-plants affected regrowth after cryopreservation. The addition of benzylaminopurine in the shooting medium had a positive effect on regrowth after liquid nitrogen exposure, while no such positive effect was observed with zeatine riboside or proline. The cryopreservation protocol established led to approximately 50% recovery with cultivar Portan and three of the four international cultivars tested. By contrast, no or very low recovery was noted with the Croatian cultivars tested. Based on ELISA tests, the GFLV virus was eliminated from 82.4% of non-cryopreserved samples and from 77.8% of cryopreserved samples in cultivar Chardonnay and the GLRaV-3 virus was eliminated from 100% of both non-cryopreserved and cryopreserved samples in cultivar Cabernet Sauvignon. These results may be related with our immunolocalisation studies, which showed that GFLV was found in the apical dome and meristematic tissues in cultivar Pinot Noir and GLRaV-3 in sieve elements of cultivar Merlot. Genetic stability of plants regenerated from cryopreserved shoot tips was studied using AFLP markers. With the eight AFLP primer combinations employed on the 43 plants tested, no polymorphism was observed after sucrose preculture, treatment with the loading solution and half-strength PVS2. However, polymorphic fragments were observed in non-cryopreserved and cryopreserved samples treated with PVS2 solution, the number of which increased with increasing durations of exposure to PVS2 solution.

grapevine; cryopreservation; cryotherapy; genetic stability

Ova disertacija je izrađena kao dvojni doktorat između Sveučilišta u Zagrebu, Agronomski fakultet i Sveučilišta u Montpellieru 2, SupAgro Montpellier